Norovirus Gastroenteritis, Carbohydrate Receptors, and
Ming Tan, Xi Jiang*
Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America
HBGAs Are an Important Factor in Norovirus
Noroviruses, an important cause of acute gastroenteritis in
humans, have been found to recognize the histo-blood group
antigens (HBGAs) as receptors. Different noroviruses revealed
different receptor-binding profiles associated with the ABO,
secretor, and Lewis HBGA types. Direct evidence of HBGA
receptor recognition in viral infection and tropism was obtained
from human volunteer challenge studies on the prototype Norwalk
virus, in which the infection rates of the volunteers matched well
with the HBGA-binding profiles of the challenge virus [1,2].
Similar evidence was also obtained from investigation of outbreaks
of gastroenteritis related to other genotypes of noroviruses [3,4],
although conflicting results also were reported. The HBGA-
binding interfaces have been identified in the protruding (P)
domain of the viral capsid protein, in which a group of scatted
amino acids forms a conformational pocket on the distal surface of
the viral capsid that interacts with individual oligosaccharide
residues of the HBGA receptors [5–7] (Figure 1). These data
indicate that the P domain is the primary site of receptor
interaction, which plays an essential role in norovirus infection.
The crystal structures of the HBGA-binding interfaces of
Norwalk virus (GI.1) and VA387 (GII.4) have been elucidated,
each representing one of the two major genogroups of human
noroviruses [5–7]. The receptor-binding interfaces of the two
strains differ significantly in their structures, precise locations,
receptor-binding modes, and amino acid compositions, although
both locate on the top of the arch-like P dimer of the viral capsids
. However, sequence alignment showed that the key residues
responsible for HBGA binding are highly conserved among strains
within but not between the two genogroups, while the remaining
sequences of the P2 subdomain are highly variable  (Figure 1).
These data indicate that HBGAs play an important role in
norovirus evolution, although other factors, such as host
immunity, may also be involved. Each of the two genogroups
represents an evolutionary lineage characterized by distinct
genetic traits. Strains within each lineage have further diverged
into sub-lineages (genotypes), probably by functional selection or
adaptation through structural constraints of the human HBGAs.
The polymorphic human HBGAs are most likely the driving force
of the divergence of human noroviruses.
Recognition of Carbohydrate Receptors May Be a
Common Feature of Caliciviruses
The initial study of a calicivirus receptor was performed on an
animal calicivirus, the rabbit hemorrhagic disease virus (RHDV)
in genus Lagovirus, which recognizes the H-type 2 HBGA . Field
surveillance and epidemiology studies showed that this recognition
is specific and associated with the resistance or susceptibility of
rabbits with or without the H-type 2 antigen to the viruses .
Following the findings of the HBGA receptors for human
noroviruses, several other caliciviruses have also been demonstrat-
ed to recognize a carbohydrate receptor. In genus Norovirus, the
bovine norovirus (GIII) was recently shown to interact with
HBGAs , while the murine norovirus (MNV, GV) recognizes
the sialic acid . In addition, the feline calicivirus (FCV) in
genus Vesivirus uses the sialic acid on the host cell surface as a
receptor, most likely for attachment . Another receptor or co-
receptor on the host cellular membrane, the junctional adhesion
molecule-1 (JAM-1), was found to be required in FCV infection,
probably helping virion penetration into host cells following the
initial attachment . Furthermore, the newly discovered rhesus
monkey calicivirus, the Tulane virus, that was isolated from
monkey stools , bound to human HBGAs .
Although further evidence for other genera of Caliciviridae,
such as Sapovirus, is needed, the available data strongly suggest that
the recognition of a carbohydrate receptor may be a common
feature of caliciviruses, even though they have adapted to different
host species after a long course of evolution. Increasing amounts of
data also showed that many bacterial and other viral pathogens
rely on a carbohydrate receptor for infection . Thus, the
requirement of a carbohydrate receptor could be a convergent
factor in the evolution of these bacterial and viral pathogens. This
principle is important not only for the research of human
noroviruses that cause acute gastroenteritis, but also for other
caliciviruses and other bacterial and viral pathogens that recognize
similar carbohydrate receptors.
Insight into the Epidemiology and Disease
Control and Prevention of Norovirus
The findings of HBGA receptors as determinants of host range
and evolution of noroviruses help our understanding of the
epidemiology of norovirus gastroenteritis. The GII.4 (genogroup
II, genotype 4) viruses have been found to predominant
everywhere in the world in the past decade. Accordingly, in vitro
binding assays revealed that most GII.4 strains recognized saliva of
Citation: Tan M, Jiang X (2010) Norovirus Gastroenteritis, Carbohydrate
Receptors, and Animal Models. PLoS Pathog 6(8): e1000983. doi:10.1371/
Editor: Hiten D. Madhani, University of California San Francisco, United States of
Published August 26, 2010
Copyright: ? 2010 Tan, Jiang. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: The authors were supported by the NIH (RO1 AI 055649, RO1 AI
037093 and PO1 HD 13021) and the Department of Defense (PR 033018) of the
USA. The funders had no role in study design,data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests
* E-mail: Jason.firstname.lastname@example.org
PLoS Pathogens | www.plospathogens.org1 August 2010 | Volume 6 | Issue 8 | e1000983
Figure 1. Elucidation of the HBGA-binding pocket and the genetic relatedness of HBGA-binding interfaces among different
genotypes of human noroviruses. The top four panels show structures of noroviruses at different levels: (from left to right) an electron
microscopy image of noroviruses, a single virus-like particle (VLP), a P dimer with indication of the carbohydrate-binding interface (colored region),
and the crystal structure of the HBGA-binding interface. The dashed square in each left panel is enlarged in the right panel. The HBGA is indicated by
a ring-shaped trisaccharide. The middle panel shows the crystal structures of the HBGA-binding interface of the prototype Norwalk virus (GI.1, left)
and the aligned sequences of the receptor-binding interface of eight GI genotypes (right). The bottom panel shows the crystal structures of the
HBGA-binding interface of strain VA387 (GII.4, left) and the aligned sequences of the receptor-binding interface of 17 GII genotypes (right). The
HBGA-binding interface can be divided into three sites representing the bottom (green) and walls (orange and red) of the binding pocket. The same
color schemes are used in the sequence alignments to highlight the conserved amino acid residues of the three sites. Partially adapted, with
permission, from .
PLoS Pathogens | www.plospathogens.org2 August 2010 | Volume 6 | Issue 8 | e1000983
all ABO secretors that represent ,80% of the general population.
This could be an important reason for the predominance of this
genotype over others that have narrower target populations. As a
result of a long period of evolution, most strains in a genotype may
have adapted to one or a few common epitopes of HBGAs. Thus,
the consensus receptor-binding profiles of individual genotypes
may not easily change. For example, a recent study showed that
the major receptor-binding property of the GII.4 viruses to H-
related antigens of secretors was traced back to a strain isolated as
early as 36 years ago . Our recent study also showed that all
major genetic clusters of GII.4 viruses isolated in the current
decade retained the consensus binding to H-related antigens ,
although changes in the HBGAs’ binding profiles among GII.4
noroviruses have also been reported . Such changes might
offer the viruses new target populations, allowing the viruses to
escape from host immunity. However, the significance of these
variants in epidemiology remains to be determined. A critical
question would be whether such variations become stable genetic
traits that replace the currently dominant strains.
The possible role of herd immunity in norovirus evolution is
another important issue for epidemiology. The surface region of
the P2 domain around the highly conserved HBGA-binding
interfaces changes significantly compared with other regions of the
capsid and other viral proteins, suggesting a potential selection
pressure from the host, such as acquired immunity. Emergence of
new dominant GII.4 variants every 2–3 years that replace the
previous ones [20,21] also suggests antigenic changes of major
circulating GII.4 strains over time. However, it is too early to
conclude whether such variants represent antigenic shift or result
in the emergence of new serotypes, as in the case of influenza
viruses. Noroviruses clearly are not spread as rapidly and
profoundly as influenza viruses because of less efficient transmis-
sion through the fecal/oral pathway compared with the respira-
tory pathway of flu. Noroviruses also may not induce a long-term
immunity to build up persistent herd immunity as quickly as flu.
Our understanding on GII.4 epidemiology and evolution is still in
the initial stages and continual studies are necessary. It is an
important issue because, if the epidemic variants represent only
minor antigenic change (drifting), the vaccine strategy of an annual
selection for flu vaccine may not be followed by a future norovirus
The findings of the conservation of the HBGA-binding
interfaces within the two major genogroups of human noroviruses
are significant for the rational design and development of antivirals
against these viruses. For example, a single compound that inhibits
the function of the highly conserved HBGA-binding pocket may
be capable of blocking infection of all strains that share the same
or similar receptor-binding interfaces. Thus, only a few com-
pounds might be sufficient to prevent infection of most human
noroviruses causing acute gastroenteritis. Furthermore, a com-
pound that is useful for the treatment of norovirus disease might
also be effective for other bacterial and viral pathogens that
recognize the same HBGA receptors.
Issues with Animal Models in Norovirus Research
Caliciviruses are known for their genetic diversity with wide host
ranges and tissue tropism, but many of them share common
carbohydrate/HBGA receptors. The role of the HBGA receptor
in viral evolution further raises the alert of zoonotic transmission of
noroviruses, because many species share common HBGA
receptors. In addition, noroviruses are highly adaptive due to a
single-stranded RNA genome, high potency of genomic recombi-
nation, and the possible quasi-species nature of the genome.
Furthermore, members of genus Norovirus that are able to infect
animals have been identified, including the bovine, murine, and
porcine noroviruses. Three genetic clusters of the porcine
noroviruses have been classified in genogroup II of human
noroviruses . Finally, an animal reservoir of human noro-
viruses has been found in oyster and other bivalve shellfishes.
Thus, further study on the origin and evolution of noroviruses and
other caliciviruses is necessary for further understanding the virus–
host interaction and potential risk of cross-species transmission of
noroviruses, which is important for disease control and prevention.
Great efforts have been made in developing an animal model
for human noroviruses. Several non-human primate species have
been challenged with human noroviruses, such as rhesus macaque,
pigtail macaque, and chimpanzee. Limited success has been
observed for clinical infection and illness in non-human primates
compared with the human host. These models are worth further
evaluation owing to their genetic and phenotypic relatedness in
many aspects with humans.
Gnotobiotic (Gn) pig is a more promising model of human
noroviruses, and currently is under investigation and development.
Pigs share several characteristics with humans in their gastroin-
testinal anatomy, physiology, immune system, and the presence of
HBGAs, such as the A and H antigens on mucosal surfaces. In a
neonatal Gn pig model, human norovirus infection has resulted in
diarrhea, virus shedding, seroconversion, immuno-cytopathic
change in the intestinal sections, and transient viremia [23–25].
Similar results have also been observed in Gn calves ,
suggesting that these Gn animal models may be useful for the
study of immunology and pathogenesis and the assessment of
vaccines and antivirals against human noroviruses.
The murine norovirus (GV)  has been used as a surrogate to
study the pathogenesis, immunology, and replication of human
noroviruses, and a great amount of data have been generated.
However, the limitations of this model are obvious due to the
difference between the two viruses in clinical manifestations
(without diarrhea/vomiting), host receptors (sialic acid versus
HBGAs), infected cell types (dendritic/macrophages versus
digestive epithelial cells), and pathogenesis. Thus, an ultimate
understanding of human noroviruses and assessment of interven-
tion approaches will most likely rely on the establishment of an
effective animal model of human noroviruses. A further animal
surrogate model may be a rhesus monkey calicivirus, the Tulane
virus. This enteric virus can replicate in vitro in monkey cell lines
. Most importantly, the Tulane virus recognizes human
HBGAs . A weakness of this model is that the Tulane virus
belongs to a unique genus separate from the Norovirus genus, and it
remains unknown whether the Tulane virus causes gastroenteritis
like human noroviruses.
Additional Questions on the Host Interaction of
As a potential key factor in co-evolution between many
microorganisms and human hosts, the polymorphic human
HBGA system may be the result of selection by some highly
virulent or life-threatening bacterial or viral pathogens in the past.
Noroviruses do not belong to these pathogens because currently
they lead only to the modest disease of acute gastroenteritis.
However, this cannot exclude the possibility that noroviruses were
once highly virulent in the past and/or may become so in the
future, because noroviruses are among those highly adaptive
species. The emergence of the highly virulent RHDV that almost
eradicated entire rabbit colonies in China and European countries
PLoS Pathogens | www.plospathogens.org3 August 2010 | Volume 6 | Issue 8 | e1000983
in the 1980s is a good example. The epidemic of SARS in 2003
could be another warning.
Noroviruses are still difficult to cultivate in vitro, even after the
discovery of HBGA receptors. One possibility is that a functional
co-receptor necessary for norovirus replication is missing in the cell
culture, although failures of additional downstream steps of viral
replication also may be the reason. In FCV, both sialic acid and
JAM-1 are required for viral replication, in which sialic acid is
believed to be a ligand or receptor for virion attachment, while the
membrane protein JAM-1 may function as a co-receptor to
facilitate FCV penetrating into the host cells. Since this two-step
process has also been shown in other viruses such as the reovirus
, and a membrane protein has been demonstrated to interact
with human noroviruses, it would be significant to explore the two-
step process to search for and characterize such a co-receptor for
The role of norovirus VP1 in interaction with host receptors has
been well studied. Little is known, however, about VP2, the minor
structural protein of the capsid. The fact that VP2 has a similar or
higher variation compared to VP1 suggests that it might also
involve a norovirus–host interaction. In addition, increasing
amounts of data showed that genomic recombination occurs
frequently among human noroviruses, with a breakpoint mainly
between the non-structural and structural genes. This would
confer recombinant variants with new genetic traits with possible
survival advantages. Finally, although human noroviruses are
highly diverse in recognition of HBGAs, only minor structural
differences in their HBGA-binding interface with shared HBGA
epitopes are expected among genetically closely related strains
(Figure 2). For example, the GII.3 viruses, such as strain MxV,
share common bindings to type A and B saliva, with only slightly
weaker binding affinities to saliva of type O secretor compared
with the consensus H binding (A, B, and O secretors) of GII.4
viruses. GII.3 has been found to predominate second only to GII.4
viruses in many countries, and GII.3 appeared to be the most
predominant genotype in the 1970s . In the laboratory a single
residue mutation around receptor-binding interfaces can result in
a change of HBGA binding patterns [8,29]. Thus, it would be of
significance to explore whether the consensus receptor binding
patterns can switch between two genotypes in nature and whether
GII.4 noroviruses will continue to dominate or will be replaced by
other genotypes in future epidemics.
Figure 2. Schematic interactions and relationships among different human noroviruses with a complete product of human HBGA.
Representative strains of different genotypes in the two major genogroups (GI and GII) of human noroviruses are shown according to their target
saccharides. Arrows indicate interactions between individual noroviruses and specific residues of human HBGAs. Dashed lines indicate a weaker
interaction. The five circles in different colors represent the five saccharide residues of a complete product of an H-related HBGA (H, A, B, Leb, or Ley).
The curved dashed arrows indicate two major binding groups, the A/B/H (blue) and the Lewis/H (black) binding groups, according to their target
residues on human HBGAs. The binding specificity and affinity of these norovirus strains were determined in .
PLoS Pathogens | www.plospathogens.org4 August 2010 | Volume 6 | Issue 8 | e1000983
1. Hutson AM, Atmar RL, Graham DY, Estes MK (2002) Norwalk virus infection
and disease is associated with ABO histo-blood group type. J Infect Dis 185:
2. Lindesmith L, Moe C, Marionneau S, Ruvoen N, Jiang X, et al. (2003) Human
susceptibility and resistance to Norwalk virus infection. Nat Med 9: 548–553.
3. Tan M, Jin M, Xie H, Duan Z, Jiang X, et al. (2008) Outbreak studies of a GII-3
and a GII-4 norovirus revealed an association between HBGA phenotypes and
viral infection. J Med Virol 80: 1296–1301.
4. Thorven M, Grahn A, Hedlund KO, Johansson H, Wahlfrid C, et al. (2005) A
homozygous nonsense mutation (428G—.A) in the human secretor (FUT2)
gene provides resistance to symptomatic norovirus (GGII) infections. J Virol 79:
5. Bu W, Mamedova A, Tan M, Xia M, Jiang X, et al. (2008) Structural basis for
the receptor binding specificity of Norwalk virus. J Virol 82: 5340–5347.
6. Cao S, Lou Z, Tan M, Chen Y, Liu Y, et al. (2007) Structural basis for the
recognition of blood group trisaccharides by norovirus. J Virol 81:
7. Choi JM, Hutson AM, Estes MK, Prasad BV (2008) Atomic resolution structural
characterization of recognition of histo-blood group antigens by Norwalk virus.
Proc Natl Acad Sci U S A 105: 9175–9180.
8. Tan M, Xia M, Chen Y, Bu W, Hegde RS, et al. (2009) Conservation of
carbohydrate binding interfaces: evidence of human HBGA selection in
norovirus evolution. PLoS ONE 4: e5058. doi:10.1371/journal.pone.0005058.
9. Ruvoen-Clouet N, Ganiere JP, Andre-Fontaine G, Blanchard D, Le Pendu J
(2000) Binding of rabbit hemorrhagic disease virus to antigens of the ABH histo-
blood group family. J Virol 74: 11950–11954.
10. Guillon P, Ruvoen-Clouet N, Le Moullac-Vaidye B, Marchandeau S, Le Pendu J
(2009) Association between expression of the H histo-blood group antigen,
alpha1,2fucosyltransferases polymorphism of wild rabbits, and sensitivity to
rabbit hemorrhagic disease virus. Glycobiology 19: 21–28.
11. Zakhour M, Ruvoen-Clouet N, Charpilienne A, Langpap B, Poncet D, et al.
(2009) The alphaGal epitope of the histo-blood group antigen family is a ligand
for bovine norovirus Newbury2 expected to prevent cross-species transmission.
PLoS Pathog 5: e1000504. doi:10.1371/journal.ppat.1000504.
12. Taube S, Perry JW, Yetming K, Patel SP, Auble H, et al. (2009) Ganglioside-
linked terminal sialic acid moieties on murine macrophages function as
attachment receptors for Murine Noroviruses (MNV). J Virol 83: 4092–4101.
13. Stuart AD, Brown TD (2007) Alpha2,6-linked sialic acid acts as a receptor for
Feline calicivirus. J Gen Virol 88: 177–186.
14. Makino A, Shimojima M, Miyazawa T, Kato K, Tohya Y, et al. (2006)
Junctional adhesion molecule 1 is a functional receptor for feline calicivirus.
J Virol 80: 4482–4490.
15. Farkas T, Sestak K, Wei C, Jiang X (2008) Characterization of a rhesus monkey
calicivirus representing a new genus of Caliciviridae. J Virol 82: 5408–5416.
16. Farkas T, Cross RW, Hargitt E 3rd, Lerche NW, Morrow AL, et al. (2010)
Genetic diversity and histo-blood group antigen interactions of rhesus enteric
caliciviruses. J Virol. In press.
17. Le Pendu J (2004) Histo-blood group antigen and human milk oligosaccharides:
genetic polymorphism and risk of infectious diseases. Adv Exp Med Biol 554:
18. Bok K, Abente EJ, Realpe-Quintero M, Mitra T, Sosnovtsev SV, et al. (2009)
Evolutionary dynamics of GII.4 noroviruses over a 34-year period. J Virol 83:
19. Yang Y, Xia M, Tan M, Huang P, Zhong W, et al. (2010) Genetic and
phenotypic characterization of GII-4 noroviruses that circulated during 1987 to
2008. J Virol 84. In press.
20. Lindesmith LC, Donaldson EF, Lobue AD, Cannon JL, Zheng DP, et al. (2008)
Mechanisms of GII.4 Norovirus Persistence in Human Populations. PLoS Med
5: e31. doi:10.1371/journal.pmed.0050031.
21. Siebenga JJ, Vennema H, Renckens B, de Bruin E, van der Veer B, et al. (2007)
Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006.
J Virol 81: 9932–9941.
22. Wang QH, Han MG, Cheetham S, Souza M, Funk JA, et al. (2005) Porcine
noroviruses related to human noroviruses. Emerg Infect Dis 11: 1874–1881.
23. Cheetham S, Souza M, McGregor R, Meulia T, Wang Q, et al. (2007) Binding
patterns of human norovirus-like particles to buccal and intestinal tissues of
gnotobiotic pigs in relation to A/H histo-blood group antigen expression. J Virol
24. Cheetham S, Souza M, Meulia T, Grimes S, Han MG, et al. (2006)
Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs. J Virol
25. Souza M, Cheetham SM, Azevedo MS, Costantini V, Saif LJ (2007) Cytokine
and antibody responses in gnotobiotic pigs after infection with human norovirus
genogroup II.4 (HS66 strain). J Virol 81: 9183–9192.
26. Souza M, Azevedo MS, Jung K, Cheetham S, Saif LJ (2008) Pathogenesis and
immune responses in gnotobiotic calves after infection with the genogroup II.4-
HS66 strain of human norovirus. J Virol 82: 1777–1786.
27. Wobus CE, Karst SM, Thackray LB, Chang KO, Sosnovtsev SV, et al. (2004)
Replication of norovirus in cell culture reveals a tropism for dendritic cells and
macrophages. PLoS Biol 2: e432. doi:10.1371/journal.pbio.0020432.
28. Barton ES, Forrest JC, Connolly JL, Chappell JD, Liu Y, et al. (2001) Junction
adhesion molecule is a receptor for reovirus. Cell 104: 441–451.
29. Tan M, Xia M, Cao S, Huang P, Farkas T, et al. (2008) Elucidation of strain-
specific interaction of a GII-4 norovirus with HBGA receptors by site-directed
mutagenesis study. Virology 379: 324–334.
30. Huang P, Farkas T, Zhong W, Tan M, Thornton S, et al. (2005) Norovirus and
histo-blood group antigens: demonstration of a wide spectrum of strain
specificities and classification of two major binding groups among multiple
binding patterns. J Virol 79: 6714–6722.
PLoS Pathogens | www.plospathogens.org5 August 2010 | Volume 6 | Issue 8 | e1000983